In 1928 Adolf Windaus was awarded the Nobel Prize in chemistry for his research on substances of significant biological importance. One of these substances was Vitamin D. Since then, much more is known about vitamin D, its chemistry and its pharmacological effects and dynamics.
Vitamin D is not widely found in food, but rather, it is produced by the skin. Once produced, it undergoes 25-hydroxylation to form a 25-hydroxy-D (25-OH-D) in the liver. The circulating concentration of 25-OH-D is considered to be an important indicator of vitamin D status in man. For example, hypovitaminosis, which results from the insufficient endogenous production of vitamin D in the skin, and insufficient dietary supplementation, and/or inability of the small intestine to absorb adequate amounts of vitamin D from dietary sources, results in hypocalcemia and hypophosphatemia and corresponding secondary hyperparathyroidism. Hypovitaminosis D also results in disturbances in mineral metabolism (i.e., rickets and osteomalacia in children and adults, respectively).
Serum 25-OH-D-levels are also found to be lower than normal in intestinal malabsorption syndromes, liver disorders (chronic and acute), and nephrotic syndromes. In osteopenia, especially in the aged, serum 25-OH-D levels are often found to be lower than normal. In cases of vitamin D intoxication, serum 25-OH-D level is found, as expected, to be higher than normal.
Once hydroxylated, 25-OH-D is again hydroxylated in the kidney to give the hormonal form 1,25-dihydroxy-vitamin D (1,25-(OH)2-D). The 1,25-(OH)2-D level in blood is also an important indicator of certain diseases. For example, a low level of 1,25-(OH)2-D is indicative of kidney failure and/or osteoporosis.
Considering their pathological importance, tremendous efforts have been directed towards developing assays for accurately measuring concentrations of 25-OH-D and/or 1,25-(OH)2-D in circulation.
For example, 25-OH-D concentrations in blood have been measured by high performance liquid chromatography (HPLC) and by competitive protein binding assays (Eisman et al., Anal. Biochem. 80: 298–305 (1977); and Haddad et al., J. Clin. Endocr. 33: 992–995 (1971)). For example, the vitamin D transport protein known as DBP, which has a strong preference for binding 25-OH-D was used in the competitive binding assay (Bouillion et al., J. Steroid Biochem. 13: 1029–1034 (1980)).
Also, various competitive binding assays have been employed to assay for the presence of 1,25-(OH)2-D. (Shigeharu et al., Anal. Biochem. 116: 211–222 (1981); Eisman et al., Arch. Biochem. Biophys. 176: 235–243 (1976); Perry et al., Biochem. Biophys. Res. Comm. 112: 431–436 (1983); Bouillion et al., Ann. Endocrin. 41: 435–436 (1980); Bouillion, Clin. Chem. 26: 562–567 (1980); Bouillion, Eur. J. Biochem. 66: 285–291 (1976)). In such assays, vitamin D and its metabolites were extracted from blood serum or plasma with an organic solvent. The extract was then purified by column chromatography and HPLC to yield 1,25-(OH)2-D. The purified 1,25-(OH)2-D was then measured.
In general, most assays presently being employed in the art to determine the concentration of vitamin D are heterogeneous assays. Furthermore, most of these assays rely on the addition of an organic solvent to release the vitamin D and/or vitamin D metabolites from the binding proteins, for example DBP. (Schmidt-Gayk, Dynamics of Bone and Cartilage Metabolism, Chapter 26, Table V). That is, the addition of organic solvent to the samples causes denaturation and precipitation of the serum proteins including DBP, and subsequently, the precipitated protein can be spun out of solution and the released metabolites remain in solution in the organic layer. This organic layer containing the released metabolite is then extracted and transferred into another system for analysis.
Although the present existing assay systems are useful, their reliance on an organic solvent to release the vitamin D and/or vitamin D metabolite is problematic. The problems with using an organic solvent include: (A) difficulties in the handling of volatile organic solvents, (B) laborious manual extractions, (C) loss of patient, identification since two transfer steps are required, and (D) loss of precision in measurement.
The loss in precision may be caused by the heterogeneous extraction step, since heterogeneous extraction can be very technique dependent. For example, in a heterogeneous extraction step, some matrix components may be extracted along with the organic layer containing the metabolite. One such matrix component is lipid, which has been shown to cause an elevated measurement of the metabolite values.
There is a need to have improved kits and methods for determining the concentration of vitamin D and its metabolites in a body fluid of a mammal.